1. Field of the Invention
The invention provides methods for distinctly detecting basepair mismatches between heteroduplex strands produced between wildtype and mutation-containing nucleic acid species. Specifically, this invention relates to methods for detecting nucleotide sequence polymorphisms including mutations in genes of biological organisms, including humans, for disease diagnosis and other purposes. More particularly, the invention relates to methods for detecting disease-related genetic polymorphisms for clinically relevant purposes, as well as providing a basic research tool for detecting genetic polymorphisms, mutations and mismatches between nucleic acid heteroduplex strands in nucleic acids of all nucleic acid-based life forms. The methods of the invention involve chemically modifying mismatched sites in such heteroduplexes and detecting the sites of chemical modification. Applications of the methods of the invention for disease detection, intervention and monitoring are also provided.
2. Background of the Related Art
In the genetic and medical arts, the existence of genetic polymorphism is frequently associated with differences between the most common species (the wildtype allele) at a genetic locus and alternative forms (the polymorphisms). When such genetic polymorphism is associated with deleterious consequences such as disease, it is frequently termed a mutation. Mutations associated with disease have been established for a number of diseases.
Genetic mutation is frequently detected in cancer, and is also the basis of profound heritable and somatic cell diseases. It is generally recognized that cancer is a multistage process involving the accumulation of mutations and other genetic alterations in a pre-neoplastic cell. The rapid and reliable detection of such altered states of nucleic acids is tantamount to defining the most significant steps of development of the resulting tumors, and has important implications for developing appropriate therapies therefor. Specifically, these mutations occur in particular growth- and differentiation-regulatory genes in eukaryotic cells, termed oncogenes and tumor suppressor genes, that are the targets for genetic lesions caused by carcinogenic chemicals, radiation and other cancer-causing substances and processes.
One of the most common types of genetic alteration in oncogenes and tumor suppressor genes is a single nucleotide substitution, termed a xe2x80x9cpoint mutation.xe2x80x9d Such point mutations typically occur against a background of the entirety of the rest of the nucleotide sequence encoding the oncogene or tumor suppressor gene. There is therefore a need in the art for the development of rapid, reliable and sensitive methods for detecting point mutations in nucleic acids (e.g., oncogenes, genetically heritable diseases) or nucleic acid changes in somatic cells, or any sequence alterations in DNA or RNA, whether wild-type DNA or DNA, or purely synthetic DNA or RNA. The method disclosed herein fulfills a critical need in the art, and is generally useful in detection, definition and diagnosis of disease. When contrasted with the current state of technology, this method accelerates the field of disease mutation discovery, with the potential of improving drug discovery for the mitigation of these diseases.
A variety of methods for detecting mutations in DNA are known in the prior art. These include direct sequencing of mutant DNA (Wong et al., 1987, Nature 195: 384-386); allele-specific oligonucleotide hybridization (Wallace et al., 1981, Nucleic Acids Res. 19: 879-895); single-strand conformation polymorphism (Orita et al., 1989, Proc. Natl. Acad. Sci. USA 86: 2766-2770); denaturing gradient gel electrophoresis (Sheffield et al., 1989, Proc. Natl. Acad. Sci. USA 86: 232-236); heteroduplex analysis (Keen et al., 1991, Trends Genet. 7: 5); and chemical (Cotton et al., 1988, Proc. Natl. Acad. Sci. USA 85: 4397-4401) or enzymatic (Youil et al., 1995, Proc. Natl. Acad. Sci. USA 92: 87-91) cleavage of mismatches. Each of these methods has serious drawbacks that preclude its use as a rapid, reliable and sensitive method for nucleic acid mismatch detection. The state of this art has recently been reviewed by Cotton (1993, Mutation Res. 285: 125-144).
The use of chemical modification of mismatches has been attempted in the prior art.
Novack et al., 1986, Proc. Natl. Acad. Sci. USA 83: 586-590 disclosed detection of a single basepair mismatch in DNA by chemical modification and gel electrophoresis.
Wani et al., 1989, Nucleic Acids Res. 17: 9957-9977 disclose immunoassays for carbodiimide-modified DNA.
Ganguly et al., 1989, Genomics 4: 530-538 disclose the use of electron microscopic methods for detecting chemically modified DNA mismatches.
Ganguly et al., 1990, Nucleic Acids Res. 18: 3933-3939 teach the use of primer extension and polymerase chain reaction to detect chemically modified DNA mismatches.
Wani et al., 1991, Biochimica et Biophysica Acta 1088: 259-269 disclose analysis of carbodiimide-modified DNA using specific antibodies as immunochemical reagents.
Zhuang et al., 1991, Amer. J. Human Genet. 48: 1186-1191 demonstrate detection of a single base mutation in a human collagen gene using direct sequencing of polymerase chain reaction-amplified, chemically modified heteroduplex DNA.
While each of these reports discloses some aspect of chemical modification of DNA to detect heteroduplexes, the prior art is devoid of teachings or disclosure of an assay having the necessary sensitivity and reliability required for routine clinical use. Moreover, the prior art, or any combination thereof, teaches only basic research applications of the technology, with the findings of these studies explicitly referenced as being xe2x80x9cinteresting.xe2x80x9d
There remains a need in the art for rapid, reliable and sensitive methods for detecting mismatches between wildtype and disease-related mutant nucleic acids, to provide genetic, clinical and other relevant (e.g., diagnostic) information for research, diagnosis and therapy.
The present invention provides methods for specifically detecting basepair mismatches between heteroduplex strands produced between reference (wildtype) and altered (mutant) nucleic acid species. The invention provides methods for chemically modifying mismatched sites in such heteroduplexes and detecting the sites of chemical modification. In preferred embodiments, chemical modification is produced using a water-soluble, single-strand DNA-specific reagent, N-cyclohexyl-Nxe2x80x2-(4-methylmorpholinium)-ethylcarbodiimide (CMC). In alternative preferred embodiments, the chemical mismatch modifying agent is detectably-labeled. In certain of these embodiments, the chemical mismatch modifying agent is provided in a xe2x80x9cmaskedxe2x80x9d state, wherein the detectable label is only detected when the chemical mismatch modifying agent is covalently linked to the site of heteroduplex mismatch. In additional alternative preferred embodiments, the chemical mismatch modifying agent further comprises a masked beacon molecule, which is unmasked upon formation of mismatch modification by the agent.
In preferred embodiments, sites of chemical mismatch modification are detected immunochemically using specific antibodies, preferably comprising polyclonal antisera and most preferably monoclonal antibodies. In alternative preferred embodiments, said antibodies are chemically linked to aggregation or accumulation agents, such as magnetic or iron beads or antigenic haptens such as biotin, to permit accumulation or aggregation of the antibodies after recognition of a chemically-modified, mismatched heteroduplex.
Kits are also provided for modifying heteroduplex nucleic acid, including reagents, buffers, single stranded nucleic acid modifying agents and enzymes such as exonuclease as described herein. Kits are also provided for detecting chemically modified heteroduplex nucleic acid, including reagents, buffers, specificity reagents such as primary and secondary antibodies, and detection reagents such as colorimetric detection reagents. Combination kits for modifying and detecting said modified heteroduplex nucleic acid are also provided by the invention.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.